Effects of dietary levels of glycine, threonine and protein on threonine efficiency and threonine dehydrogenase activity in hepatic mitochondria of chicks.

This study was carried out to evaluate the relationship between threonine (Thr) efficiency and Thr dehydrogenase (TDG) activity as an indicator of Thr oxidation on chicks fed with levels of diets (CP [17.5% and 21.5%] and Thr [3.8 and 4.7 g/100 g CP]; glycine [Gly][0.64% and 0.98%] and true digestible Thr [dThr] [0.45% and 0.60%]). Calculation of the Thr efficiency was based on N-balance data and an exponential N-utilization model, and TDG activity was determined as accumulation of aminoacetone and Gly during incubation of hepatic mitochondria. This study found that in the liver of chicks who received a diet containing up to 0.79% Thr (4.7 g Thr/100 g of CP) in the 17.5% CP diet, no significant (p>0.05) effect on TDG activity was observed. However, significantly (p = 0.014) increased TDG activity was observed with a diet containing 21.5% CP (4.7 g Thr/100 g of CP) and the efficiency of Thr utilization showed a significant (p = 0.001) decrease, indicating the end of the Thr limiting range. No significant (p>0.05) effect on the total TDG activity and accumulation of Gly was observed with addition of Gly to a diet containing 0.45% dThr. In addition, addition of Gly to a diet containing 0.60% dThr also did not result in a change in accumulation of Gly. Due to an increase in accumulation of aminoacetone, an elevated effect on total TDG activity was also observed. No significant (p>0.05) reduction in the efficiency of Thr utilization was observed after addition of Gly at the level of 0.45% dThr. However, significantly (p<0.001) reduced efficiency of Thr utilization was observed after addition of Gly at the level of 0.60% dThr. Collectively, we found that TDG was stimulated not only by addition of Thr and protein to the diet, but also by addition of Gly, and efficiency of Thr utilization was favorably affected by addition of Gly at the level near to the optimal Thr concentration. In addition, no metabolic requirement of Gly through the TDG pathway was observed with almost the same accumulation of Gly and a slight increase in TDG activity by addition of Gly. Thus, our findings suggest that determination of TDG activity and parameter of efficiency of Thr utilization may be useful for evaluation of dietary Thr level.

Nuclear localization of beta-catenin is an important prognostic factor in hepatoblastoma.

In this study, mutational and immunohistochemical analyses of beta-catenin were performed in 30 hepatoblastomas, to assess the prevalence of alterations of the Wnt pathway with respect to clinicopathological parameters and survival. Four missense mutations of beta-catenin (13.3%) were detected and there was strong immunoreactivity for beta-catenin in the cytoplasm and/or the nucleus in 97% of hepatoblastomas. Nuclear and cytoplasmic staining was demonstrated in 19 of 30 tumours (63%), while ten revealed only cytoplasmic staining. Statistically, this nuclear beta-catenin staining was significantly higher in the embryonal (Fisher exact test; p=0.00393) or undifferentiated type (p=0.00156) of hepatoblastoma than in the fetal type, but there was no difference between clinical stages I and II and clinical stages III and IV (p=0.175). Cumulative survival curves showed that nuclear beta-catenin staining (generalized Wilcoxon test; p=0.0088), undifferentiated histological type (p=0.0305), and clinical stages III and IV (p=0.0107) were significantly correlated with shorter survival time in these patients. Moreover, Cox multivariate analysis provides evidence that nuclear beta-catenin staining is the most important prognostic factor for survival (p=0.0090). It is therefore concluded that immunohistochemical analysis of beta-catenin might be a useful clinical tool for estimating the prognosis for patients with hepatoblastoma.

Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription.

Beta-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets beta-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of beta-catenin in melanoma cells expressing proteasome-resistant beta-catenin, as well as the effects that perturbation of beta-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity. Beta-catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with beta-catenin without perturbing either LAR or beta-catenin levels or LAR/beta-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of beta-catenin and increased beta-catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclear beta-catenin level and inhibited beta-catenin-driven transcription, as assessed using two different beta-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two beta-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the beta-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant beta-catenin is regulated further by geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.

Mapping of a new target region of allelic loss at 21q22 in primary gastric cancers.

To determine the minimal region of deletion on 21q22 in gastric cancer, we performed a high-density loss of heterozygosity (LOH) study with eight polymorphic microsatellite markers. Among the 43 tumors examined, 20 (50%) of 40 informative carcinomas showed LOH at one or more loci. The peak LOH frequency was identified at D21S1820 (34.2%) in 21q22.3. This data suggests that this locus might harbor a new tumor suppressor gene in an area <0.332 Mb in physical map distance defined by D21S1820 and D21S49. Thus, we speculate that trefoil factor family 1 (TFF1), located in this narrow region, might be the most probable candidate gene involved in gastric cancer carcinogenesis.

Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G(1) phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex.

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimeric bcr-abl gene giving rise to a p210(Bcr-Abl) protein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G(1) phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210(Bcr-Abl), Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle-dependent kinases 4 and 6 and up-regulated cell cycle-dependent kinase inhibitor p27(Kip1) protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210(Bcr-Abl) formed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210(Bcr-Abl) from Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210(Bcr-Abl).

The heat shock protein 90 antagonist geldanamycin alters chaperone association with p210bcr-abl and v-src proteins before their degradation by the proteasome.

Several important signaling proteins including transcription factors and protein kinases depend on heat shock protein (Hsp)-90 for stability. p210bcr-abl, a protein expressed in chronic myelogenous leukemia, is functionally inhibited by the benzoquinone ansamycin herbimycin A. Benzoquinone ansamycins also bind to and inhibit the activity of Hsp90. We now demonstrate that p210bcr-abl is complexed with Hsp90 and its cochaperone p23 in K562 chronic myelogenous leukemia cells. Brief exposure to the benzoquinone ansamycin Hsp90 inhibitor geldanamycin (GA) decreases the association of p210bcr-abl with Hsp90 and p23 and increases its association with the chaperones Hsp70 and p60Hop. GA has a similar effect on chaperone association with v-src, another Hsp90-dependent oncogenic kinase. Loss of Hsp90/p23 association and acquisition of Hsp70/p60Hop association of both p210bcr-abl and v-src precede GA-induced degradation of these kinases. GA-induced degradation is mediated by the proteasome because proteasome inhibitors block the effects of GA, causing both p210bcr-abl and v-src to accumulate in a detergent-insoluble cellular fraction. Both p210bcr-abl and v-src are more susceptible to GA-induced degradation than are their normal cellular counterparts, c-abl and c-src.

Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition.

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.

Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis.

Little is known about the molecular mechanisms that control adrenomedullin (AM) production in human cancers. We demonstrate here that the expression of AM mRNA in a variety of human tumor cell lines is highly induced in a time-dependent manner by reduced oxygen tension (1% O2) or exposure to hypoxia mimetics such as desferrioxamine mesylate (DFX) or CoCl2. This AM expression seems to be under hypoxia-inducible factor-1 (HIF-1) transcriptional regulation, since HIF-1alpha and HIF-1beta knockout mouse cell lines had an ablated or greatly reduced hypoxia AM mRNA induction. Similarly, inhibition or enhancement of HIF-1 activity in human tumor cells showed an analogous modulation of AM mRNA. Under hypoxic conditions, immunohistochemical analysis of tumor cell lines revealed elevated levels of AM and HIF-1alpha as compared with normoxia, and we also found an increase of immunoreactive AM in the conditioned medium of tumor cells analyzed by RIA. AM mRNA stabilization was shown to be partially responsible for the hypoxic up-regulated expression of AM. In addition, we have identified several putative hypoxia response elements (HREs) in the human AM gene, and reporter studies with selected HREs were capable of enhancing luciferase expression after exposure to DFX. Furthermore, transient coexpression of HIF-1alpha resulted in an augmented transactivation of the reporter gene after DFX treatment. Given that most solid human tumors have focal hypoxic areas and that AM functions as a mitogen, angiogenic factor, and apoptosis-survival factor, our findings implicate the HIF-1/AM link as a possible promotion mechanism of carcinogenesis.

Regulation of BRCA1 by protein degradation.

BRCA1, a tumor suppressor protein implicated in hereditary forms of breast and ovarian cancer, is transcriptionally regulated in a proliferation-dependent manner. In this study, we demonstrate a substantial role for proteolysis in regulating the BRCA1 steady-state protein level in several cell lines. N-acetyl-leu-leu-norleucinal (ALLN), an inhibitor of the proteasome, calpain, and cathepsins, caused BRCA1 protein to accumulate in the nucleus of several human breast, prostate, and melanoma cell lines which express low or undetectable basal levels of BRCA1 protein, but not in cells with high basal expression of BRCA1. Protease inhibition did not increase BRCA1 synthesis, nor change its mRNA level, but it dramatically prolonged the protein's half-life. In contrast to ALLN, lactacystin and PS341, two specific proteasome inhibitors, as well as calpastatin peptide and PD150606, two selective calpain inhibitors, had no effect on BRCA1 stability, whereas ALLM, an effective calpain and cathepsin inhibitor but weak proteasome inhibitor, did stimulate accumulation of BRCA1. Moreover, three inhibitors of acidic cysteine proteases, chloroquine, ammonium chloride and bafilomycin, were as effective as ALLN. These results demonstrate that degradation by a cathepsin-like protease in fine balance with BRCA1 transcription is responsible for maintaining the low steady-state level of BRCA1 protein seen in many cancer cells.

Nickel-induced transformation shifts the balance between HIF-1 and p53 transcription factors.

Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. In an attempt to unravel the molecular mechanisms of Ni-induced transformation we investigated transcriptional activity of hypoxia-inducible factor (HIF-1) and p53 tumor suppressor protein in Ni-transformed cells. We demonstrated that the activity of HIF-1-responsive promoters was increased in Ni-transformed rodent cells resulting in the increased ratio between HIF-1- and p53-stimulated transcription. To further elucidate the roles of HIF-1 and p53 in Ni-induced transformation we used human osteosarcoma (HOS) cells and a Ni-transformed derivative, SA-8 cells. Since non-functional p53 was expressed in both HOS and SA-8 cells, acute Ni treatment induced HIF-1alpha protein and HIF-1-dependent transcription without affecting p53. In MCF-7 and A549, human cancer cells with the wild-type p53, both functional p53 and HIF-1alpha proteins accumulated following exposure to Ni. The induction of HIF-1alpha and wild-type p53 by Ni was detected after 6 h and was most pronounced by 24 h. These results suggest that acute Ni treatment causes accumulation of HIF-1alpha protein and simultaneous accumulation of wild-type, but not mutant, p53. We suggest that the induction of hypoxia-like conditions in Ni-treated cells with subsequent selection for increased HIF-1-dependent transcription is involved in Ni-induced carcinogenesis.

Inhibitors of transcription, proteasome inhibitors, and DNA-damaging drugs differentially affect feedback of p53 degradation.

Mutations of the p53 gene are the most common abnormalities in human cancer. In contrast to mutant p53, wild-type (wt) p53 protein is present at low levels due to rapid degradation by proteasome. We demonstrated that wt p53 protein stabilization following DNA damage or proteasome inhibition did not abolish the wild-type conformation. DNA damage did not cause accumulation of ubiquitinated forms of wt p53, suggesting abrogation of ubiquitination. Consistent with this, the E6 oncoprotein which targets p53 for ubiquitination abolished stabilization of p53 protein by DNA-damaging drugs but not by proteasome inhibitors. In contrast to the effects on wt p53, inhibitors of proteolysis downregulated mutant p53. Regulation of p53 levels can be explained by a feedback mechanism where wt p53 transcriptionally induces "sensor" proteins (Mdm-2, as an example) and these, in turn, target p53 for degradation. Like p53, Mdm-2 is degraded by proteasome. Therefore, inhibition of proteasome caused accumulation of Mdm-2, leading to degradation of mutant p53 by the remaining proteolytic activity of the cell. We propose that inhibition of transcription should increase wt p53 protein due to inhibition of Mdm-2 synthesis. An inhibitor of transcription, alpha-amanitin, dramatically induced wt p53 protein, whereas Mdm-2 protein was downregulated. Moreover, alpha-amanitin increased p53 protein levels in E6-transfected cells. Although inhibitors of transcription, such as actinomycin D, also damage DNA, reduction of Mdm-2 or other putative "sensor" proteins may contribute to their p53-stabilizing activity. Similarly, antimetabolites augment accumulation of wt p53 due to interference with RNA synthesis.

p53 inhibits hypoxia-inducible factor-stimulated transcription.

p53 is required for hypoxia-induced apoptosis in vivo, although the mechanism by which this occurs is not known. Conversely, induction of the hypoxia-inducible factor-1 (HIF-1) transactivator stimulates transcription of a number of genes crucial to survival of the hypoxic state. Here we demonstrate that p53 represses HIF-1-stimulated transcription. Although higher levels of p53 are required to inhibit HIF than are necessary to transcriptionally activate p53 target genes, these levels of p53 are similar to those that stimulate cleavage of poly(ADP-ribose) polymerase, an early event in apoptosis. Transfection of full-length p300 stimulates both p53-dependent and HIF-dependent transcription but does not relieve p53-mediated inhibition of HIF. In contrast, a p300 fragment, which binds to p53 but not to HIF-1, prevents p53-dependent repression of HIF activity. Transcriptionally inactive p53, mutated in its DNA binding domain, retains the ability to block HIF transactivating activity, whereas a transcriptionally inactive double point mutant defective for p300 binding does not inhibit HIF. Finally, depletion of doxorubicin-induced endogenous p53 by E6 protein attenuates doxorubicin-stimulated inhibition of HIF, suggesting that a p53 level sufficient for HIF inhibition can be achieved in vivo. These data support a model in which stoichiometric binding of p53 to a HIF/p300 transcriptional complex mediates inhibition of HIF activity.

Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha.

Although hypoxia (lack of oxygen in body tissues) is perhaps the most physiological inducer of the wild-type p53 gene, the mechanism of this induction is unknown. Cells may detect low oxygen levels through a haem-containing sensor protein. The hypoxic state can be mimicked by using cobalt chloride and the iron chelator desferrioxamine: like hypoxia, cobalt chloride and desferrioxamine activate hypoxia-inducible factor 1alpha (HIF-1alpha), which stimulates the transcription of several genes that are associated with hypoxia. Here we show that these treatments induce accumulation of wild-type p53 through HIF-1alpha-dependent stabilization of p53 protein. Induction of p53 does not occur in either a mutant hepatoma cell line that is unable to induce HIF-1alpha or embryonic stem cells derived from mice lacking HIF-1beta. HIF-1alpha is found in p53 immunoprecipitates from MCF7 cells that express wild-type p53 and are either hypoxic or have been exposed to desferrioxamine. Similarly, anti-haemagglutinin immunoprecipitates from lysates of normoxic PC3M cells that had been co-transfected with haemagglutinin-tagged HIF-1alpha and wild-type p53 also contain p53. Transfection of normoxic MCF7 cells with HIF-1alpha stimulates a co-transfected p53-dependent reporter plasmid and increases the amount of endogenous p53. Our results suggest that hypoxic induction of transcriptionally active wild-type p53 is achieved as a result of the stabilization of p53 by its association with HIF-1alpha.

Geldanamycin-induced destabilization of Raf-1 involves the proteasome.

The Raf-1-MEK-MAPK pathway plays an important role in transducing extracellular growth factor signaling into altered nuclear transcription factor function. The benzoquinone ansamycin Geldanamycin (GA) specifically binds to the heat shock protein HSP90 and alters its complex with Raf-1. This leads to a decrease in Raf-1 levels and to disruption of the Raf-1-MEK-MAPK signaling pathway. The enhanced degradation of Raf-1 protein was prevented by inhibitors of the proteasome, while inhibition of lysosomal or other proteases was ineffective. Raf-1 that was protected from GA-induced degradation was of higher molecular weight and showed a laddering pattern consistent with its polyubiquitination. Unlike Raf-1 in untreated cells, the protein was insoluble in Triton X100- or NP40-based buffers. Signaling through this pathway was inhibited by GA, concomitant with loss of Raf-1 protein, but was restored if Raf-1 was protected from GA-induced degradation by proteasome inhibitors.

Geldanamycin-stimulated destabilization of mutated p53 is mediated by the proteasome in vivo.

Mutation of the tumor suppressor gene p53 is the most common genetic abnormality detected in human cancers. Wild type p53 is a short-lived protein with very low basal intracellular levels. Most mutated forms of the protein, however, display markedly increased intracellular levels as an essential feature of their positive transforming activity. In this report, we have used selective inhibitors of the 20S proteasome to demonstrate that processing of p53 by ubiquitination and proteasome-mediated degradation is impaired by commonly occuring mutations of the protein. We found that this impairment of p53 turnover can be reversed by treatment of tumor cells with the benzoquinone ansamycin, geldanamycin, leading to a marked reduction in intracellular p53 levels. Finally, using cells which over-express a mutant p53 protein, we were able to demonstrate that restoration of proteasome-mediated degradation by geldanamycin is accompanied by p53 polyubiquitination. Although much remains to be learned about the mechanisms involved, our data demonstrate that selective de-stabilization of mutant transforming proteins such as p53 can be achieved pharmacologically with agents such as geldanamycin which modify the function of molecular chaperone proteins within tumor cells.

Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity.

PURPOSE: Recently, it has been shown that geldanamycin (GA), a benzoquinone ansamycin, is able to deplete mutant p53, p185erbB2 and Raf-1 proteins in cancer cells. However, the relationship between these activities of GA and its antiproliferative activity is not clear. Here we investigated the effects of 28 GA derivatives in SKBr3, a human breast cancer cell line. METHODS: We performed Western blot analysis of Raf-1, p185erbB2 and mutant p53 proteins following drug treatment and correlated these findings with the cytotoxicity of the various GA derivatives. RESULTS: We found that downregulation of Raf-1, p185erbB2 and mutant p53 proteins was correlated. Thus, a drug that was active against one oncoprotein was equally active against the two others. Inactive derivatives were identified by their inability to downregulate these oncoproteins, even at a high dose (2 microM). These inactive drugs also had no or minimal antiproliferative activity (IC50 > 3 microM). All other analogs (at a concentration of 2 microM) downregulated p53, p185erbB2, and Raf-1, and also displayed cytotoxicity (IC50 in the range 6-600 nM). This category of drugs was further divided into more- and less-active agents by testing at lower doses (40 nM). The drugs that remained active against their molecular targets had an IC50 for antiproliferative activity of less than 40 nM. Maximal effects on mutant p53, p185erbB2 and Raf-1 were observed at doses that were 4-5 times greater than the cytotoxic IC50. CONCLUSIONS: These findings suggest that GA and its derivatives are cytostatic/cytotoxic at concentrations that also downregulate Raf-1, p185erbB2 and mutant p53, and raise the possibility that depletion of these proteins and the antiproliferative activities of GA have a common mechanism.